The long-term goal of this project is to characterize the mechanisms that regulate apoptosis (programmed cell death, or PCD) and restrict it to the appropriate cells in C. elegans. Apoptosis in C. elegans is triggered by CED-3 (a caspase), which requires CED-4 (an Apaf-1 family member) for its activation. Two C. elegans genes, icd-1 and -2, identified in this laboratory, result in widespread CED-4-dependent, CED-3-independent apoptosis. A comprehensive set of experiments in C. elegans and human cells will be performed to illuminate the action of ICD-1 and -2 in repression of this CED-4-mediated apoptosis. In the first aim, the specificity of the CED-4-dependent PCD and sterility will be investigated in chromosomal mutants lacking icd gene activity and the hypothesis that icd-1 and icd-2 are functionally separable will be tested. The potential conservation of their function will be examined by analyzing whether they can inhibit apoptosis in human cells and whether the human proteins can rescue the C. elegans mutants. In the second aim, a model accounting for the action of ICD-1 and -2 in the core apoptotic pathway will be critically evaluated by analyzing their fate, mechanism of action, and interactions in the PCD pathway. The hypotheses that ICD-1 is a substrate of caspases, that ICD proteins can inhibit caspase activity, and that a conserved caspase cleavage site and caspase recruitment domain (CARD) participate in the PCD-repressing function of ICD-1 in dying cells will be tested. Further, the role of other caspases in the CED-3-independent, CED-4-dependent PCD seen in icd(-) mutants and the interactions between ICD-1, ICD-2, and PCD pathway components will be examined. Finally, new apoptotic regulators (some which inhibit apoptosis in mammals) identified by RNAi-based screens will be analyzed. These genes were identified based on CED-3-independent apoptosis or CED-4-dependent sterility when their activity is diminished. The genes will be categorized into phenotypic and functional groups and the action of selected genes in the PCD pathway investigated. These studies should help to advance our understanding of both apoptotic regulation and the underlying causes of many diseases that result from dysregulated apoptosis by characterizing candidate factors that may be dysfunctional in human neurodegenerative diseases or that are inappropriately activated in cancers. [unreadable] [unreadable]